This Hsp70 dose response assay is developed and performed for the purpose of SAR study on analogs of hits originally identified in the fluorescence polarization HTS assay for Hsp70 Inhibitors (AID 583). ..more

This Hsp70 dose response assay is developed and performed for the purpose of SAR study on analogs of hits originally identified in the fluorescence polarization HTS assay for Hsp70 Inhibitors (AID 583).

Over-expression of molecular chaperones occurs commonly in cancers and provides protection from a wide variety of cellular stresses, both endogenous and iatrogenic. Molecular chaperones also play important roles in maintaining the activity of several signal-transducing proteins and transcriptions factors involved in malignant transformation. The human genome contains nine Hsp70-family genes. These chaperones include Hsp70 and Hsc70, which are commonly over-expressed in cancers and which confer resistance to myriad cellular stresses, including cytotoxic chemotherapy.

A positive of the assay is defined as a compound with IC50 value in the range of tested concentrations, i.e. below 100 uM. Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics were devised to take into consideration compound efficacy, the screening stage of the data and apparent compound behavior in the assay. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the Hsp70 assay is as follows: 1) First tier (0-40 range) is reserved for primary screening data and therefore is not applicable in this assay. 2) Second tier (41-80 range) is reserved for dose-response confirmation data of the primary hits and therefore is not applicable in this assay.3) Third tier (81-100 range) is reserved for dry-powder compounds that represent purchased and resynthesized positives and their analogues and utilized for SAR studies. a. Compounds that failed to reproduce from dry powder or have IC50 > 100 uM are assigned inactive and a score value of 81. b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC: QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]This empirical factor prorates the likelihood of a target- or a pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on the expectation that a compound with a single mode of action that achieved an equilibrium in the assay would demonstrate the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their divergence.c. The score is calculated using the following equation: Score = 82+3*(pIC50-4)*QC, where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units, and QC is calculated using Hill coefficient as above. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior in the assays.